A study of developmentally-regulated mRNA populations in Physarum polycephalum
Methods for the isolation of pure, intact Physarum polysomal RNA have been employed to obtain polyadenylated RNA. Physarum polyadenylated RNA isolated by these methods has been demonstrated to be suitable for use as a template for the synthesis of Physarum single-stranded cDNA. These molecules ranged in size from 100--2000 nucleotides. The enzyme steps involved in the synthesis and cloning of double-stranded cDNA were investigated individually to determine the optimum reaction conditions. A globin mRNA template was employed to analyse conditions for single and double-stranded cDNA synthesis. It was demonstrated that synthesis of the second cDNA strand, S1 Nuclease treatment, and synthesis of homopolymer tracts are inefficient and variable features of the standard cDNA protocols employed at the time of the study. These difficulties have been reported by other workers, and it has been suggested as a result that alternative methods of cDNA synthesis and cloning should be employed. Several cDNA clones, obtained from a limited Physarum cDNA library, were analysed in detail. One, pPCF2, was demonstrated to hybridize to methylated, repetitive Physarum DNA by Southern hybridization. Therefore in this instance, methylation of CpG sequences, which has been implicated in the negative regulation of gene expression, did not inhibit transcription of DNA containing these sequences, as has been proposed. The DNA sequence which hybridized to pPCF2 contained internal, methylated sequences, and was not methylated at the flanking restriction endonuclease recognition sites. This is consistent with observations that methylation at the 5' promoter region of the gene, inhibits expression of that gene, while methylation of the structural region does not. Another Physarum cDNA clone, pPCF3, was demonstrated to hybridize exclusively to undermethylated, repetitive Physarum DNA sequences. A hypermethylated, highly repetitive, cloned Physarum DNA sequence, pPH29, was shown to hybridize to cytoplasmic and polysomal RNA. Recently, pPH29 has been identified as the middle sequence of a putative transposon-like element. Such elements are believed to transpose via an RNA intermediate, which is reverse-transcribed into cDNA. The demonstration that pPH29 hybridizes to Physarum RNA provides further evidence, in conjunction with structural information, that it may form part of a transposable element.